Validation of presumptive tests for non-human blood using Kastle Meyer and Hemastix reagents.

Kastle Meyer and Hemastix reagents are presumptive tests commonly used in forensic casework for the detection of blood, and their suitability has been reviewed in numerous publications. However, studies to date have focused on the validation of these tests on human blood alone, and no published work has looked at the sensitivity, specificity and effect on DNA analysis when using these reagents to presumptively test for animal blood. The aim of this study was to validate the two reagents for use with animal blood, and compare their performance in order to choose the best test based on the circumstances in wildlife crime investigation. The sensitivity, specificity, stability and robustness of the methods were assessed by experiments with dilutions of animal blood (from 1:4 to 1:65536) using direct and indirect (rub) tests, potential interfering substances, blood sources from different species and aged blood. The effects of the two reagents on subsequent DNA analysis were also investigated. During the direct tests, Kastle Meyer showed a higher sensitivity, detecting blood down to a dilution of 1:16,384, one order of magnitude lower than Hemastix. However during the rub test, Hemastix showed a higher sensitivity, detecting blood down to a dilution of 1:64 on porous materials while Kastle Meyer was positive only down to a dilution of 1:16. Moreover, when using the same swab for presumptive testing and DNA extraction, Hemastix testing allowed amplification of a sufficient amount of DNA for species identification at its limit of sensitivity on porous materials (1:64) while Kastle Meyer inhibited most amplification of DNA at its less sensitive limit of 1:16 dilution. On the other hand, Hemastix showed a much lower specificity, producing false positive results when exposed to tomato, potato, rust, avian uric acid, bleach and sink rot, while Kastle Meyer only produced a faint positive reaction from potato. Both tests performed equally well detecting fresh blood of different animal species. The stability test gave comparable results among the tests except for aged fish blood stains, where the Kastle Meyer test performed poorly. Owing to its ease of use, higher sensitivity, and lack of interference with downstream DNA analysis, and despite its reduced specificity compared to Kastle Meyer, the Hemastix method is more appropriate for use in wildlife crime investigations. Positive results would always be confirmed with DNA analysis and the low interference of the reagent will allow the use of a single swab for presumptive testing and DNA sampling.

Profiling in wildlife crime: Recovery of human DNA deposited outside.

Incidents of bird of prey persecution receive a lot of media coverage in the UK, with investigations rarely recovering sufficient evidence to proceed to prosecution. One of the main challenges is to identify a suspect, as these offences are carried out in remote locations without witnesses, and crime scenes may not be found for days. However, traps, poisoned baits and bird of prey carcasses can be recovered from these crime scenes. This study aimed to determine whether reportable human DNA profiles could be recovered from any of these substrates after periods of time outside. Experiments depositing human touch DNA on duplicate substrates (traps, rabbit baits and corvid carcasses) set for 0, 1, 2, 4, 7 and 10 days outside were carried out, with DNA recovery and profiling following standard operating procedures for Scottish Police Authority Forensic Services. Weather conditions varied among experiments, including some heavy rainfall. Results demonstrated that it was possible to obtain reportable DNA profiles from all substrates after at least 1 day outside. Most promisingly, the traps showed no drop-off in DNA persistence over the experiments as complete DNA profiles were obtained after the full 10 days outside. A further experiment using 4 bird of prey carcasses confirmed that it is possible to obtain reportable human DNA profiles from them after 1 day outside (n = 2 reportable profiles). These results show that touch DNA can persist in an outdoor environment, and provide a tantalising avenue for inquiry in bird of prey persecution investigations.

Performance and precision of double digestion RAD (ddRAD) genotyping in large multiplexed datasets of marine fish species.

The development of Genotyping-By-Sequencing (GBS) technologies enables cost-effective analysis of large numbers of Single Nucleotide Polymorphisms (SNPs), especially in "non-model" species. Nevertheless, as such technologies enter a mature phase, biases and errors inherent to GBS are becoming evident. Here, we evaluated the performance of double digest Restriction enzyme Associated DNA (ddRAD) sequencing in SNP genotyping studies including high number of samples. Datasets of sequence data were generated from three marine teleost species (>5500 samples, >2.5 × 1012 bases in total), using a standardized protocol. A common bioinformatics pipeline based on STACKS was established, with and without the use of a reference genome. We performed analyses throughout the production and analysis of ddRAD data in order to explore (i) the loss of information due to heterogeneous raw read number across samples; (ii) the discrepancy between expected and observed tag length and coverage; (iii) the performances of reference based vs. de novo approaches; (iv) the sources of potential genotyping errors of the library preparation/bioinformatics protocol, by comparing technical replicates. Our results showed use of a reference genome and a posteriori genotype correction improved genotyping precision. Individual read coverage was a key variable for reproducibility; variance in sequencing depth between loci in the same individual was also identified as an important factor and found to correlate to tag length. A comparison of downstream analysis carried out with ddRAD vs single SNP allele specific assay genotypes provided information about the levels of genotyping imprecision that can have a significant impact on allele frequency estimations and population assignment. The results and insights presented here will help to select and improve approaches to the analysis of large datasets based on RAD-like methodologies.

Identification of genes responding to nematode infection in red grouse.

The identification of genes involved in a host's response to parasite infection provides both a means for understanding the pathways involved in immune defence and a target for examining host-parasite co-evolution. Most studies rely on a candidate gene approach derived from model systems to identify gene targets of interest, and there have been a dearth of studies geared towards providing a holistic overview of immune response from natural populations. We carried out an experiment in a natural population of red grouse (Lagopus lagopus scoticus) to manipulate levels of Trichostrongylus tenuis parasite infection. The transcriptomic response of individuals was examined from standard cDNA and suppressive subtractive hybridization (SSH) libraries produced from gut, liver and spleen, enriching for genes expressed in response to T. tenuis infection. A total of 2209 and 3716 unique transcript sequences were identified from the cDNA and SSH libraries, respectively. Forty-five of these had Gene Ontology annotation associated with immune response. Some of these genes have previously been reported from laboratory-based studies of model species as important in immune response to gastrointestinal parasite infection; however, multiple novel genes were also identified. These may reveal novel pathways involved in the host response of grouse to T. tenuis and provide a resource that can be utilized as candidate genes in other species. All sequences described have been deposited in GenBank (accession numbers GW698221-GW706922)

Transcriptomic response of red grouse to gastro-intestinal nematode parasites and testosterone: implications for population dynamics.

A central issue in ecology is in understanding the relative influences of intrinsic and extrinsic effects on population regulation. Previous studies on the cyclic population dynamics of red grouse (Lagopus lagopus scoticus) have emphasized the destabilizing effects of either nematode parasites or territorial behaviour and aggression. The potential interacting effects of these processes, mediated through density-dependent, environmentally induced alterations of host immunocompetence influencing susceptibility to parasites have not been considered. Male red grouse at high density are more aggressive, associated with increased testosterone, which potentially could lead to reduced immunocompetence at a stage when parasites are most prevalent. This could depress individual condition, breeding performance and survival and thus drive or contribute to overall reductions in population size. Here, we characterize the transcriptomic response of grouse to nematode parasite infection and investigate how this is subsequently affected by testosterone, using a microarray approach contrasting red grouse with high and low parasite load at both high and low testosterone titre. A suite of 52 transcripts showed a significant level of up-regulation to either chronic parasite load or experimental parasite infection. Of these, 51 (98%) showed a reduced level of expression under conditions of high parasite load and high testosterone. The genes up-regulated by parasites and then down-regulated at high testosterone titre were not necessarily associated with immune response, as might be intuitively expected. The results are discussed in relation to the fitness and condition of individual red grouse and factors influencing the regulation of abundance in natural populations.

Testing the interactive effects of testosterone and parasites on carotenoid-based ornamentation in a wild bird.

Testosterone underlies the expression of most secondary sexual traits, playing a key role in sexual selection. However, high levels might be associated with physiological costs, such as immunosuppression. Immunostimulant carotenoids underpin the expression of many red-yellow ornaments, but are regulated by testosterone and constrained by parasites. We manipulated testosterone and nematode burdens in red grouse (Lagopus lagopus scoticus) in two populations to tease apart their effects on carotenoid levels, ornament size and colouration in three time-step periods. We found no evidence for interactive effects of testosterone and parasites on ornament size and colouration. We showed that ornament colouration was testosterone-driven. However, parasites decreased comb size with a time delay and testosterone increased carotenoid levels in one of the populations. This suggests that environmental context plays a key role in determining how individuals resolve the trade-off between allocating carotenoids for ornamental coloration or for self-maintenance needs. Our study advocates that adequately testing the mechanisms behind the production or maintenance of secondary sexual characters has to take into account the dynamics of sexual trait expression and their environmental context.

Oxidative stress and the effect of parasites on a carotenoid-based ornament.

Oxidative stress, the physiological condition whereby the production of reactive oxygen and nitrogen species overwhelms the capacity of antioxidant defences, causes damage to key bio-molecules. It has been implicated in many diseases, and is proposed as a reliable currency in the trade-off between individual health and ornamentation. Whether oxidative stress mediates the expression of carotenoid-based signals, which are among the commonest signals of many birds, fish and reptiles, remains controversial. In the present study, we explored interactions between parasites, oxidative stress and the carotenoid-based ornamentation of red grouse Lagopus lagopus scoticus. We tested whether removing nematode parasites influenced both oxidative balance (levels of oxidative damage and circulating antioxidant defences) and carotenoid-based ornamentation. At the treatment group level, parasite purging enhanced the size and colouration of ornaments but did not significantly affect circulating carotenoids, antioxidant defences or oxidative damage. However, relative changes in these traits among individuals indicated that males with a greater number of parasites prior to treatment (parasite purging) showed a greater increase in the levels of circulating carotenoids and antioxidants, and a greater decrease in oxidative damage, than those with initially fewer parasites. At the individual level, a greater increase in carotenoid pigmentation was associated with a greater reduction in oxidative damage. Therefore, an individual's ability to express a carotenoid-based ornament appeared to be linked to its current oxidative balance and susceptibility to oxidative stress. Our experimental results suggest that oxidative stress can mediate the impact of parasites on carotenoid-based signals, and we discuss possible mechanisms linking carotenoid-based ornaments to oxidative stress.

Physiological stress links parasites to carotenoid-based colour signals.

Vertebrates commonly use carotenoid-based traits as social signals. These can reliably advertise current nutritional status and health because carotenoids must be acquired through the diet and their allocation to ornaments is traded-off against other self-maintenance needs. We propose that the coloration more generally reveals an individual's ability to cope with stressful conditions. We tested this idea by manipulating the nematode parasite infection in free-living red grouse (Lagopus lagopus scoticus) and examining the effects on body mass, carotenoid-based coloration of a main social signal and the amount of corticosterone deposited in feathers grown during the experiment. We show that parasites increase stress and reduce carotenoid-based coloration, and that the impact of parasites on coloration was associated with changes in corticosterone, more than changes in body mass. Carotenoid-based coloration appears linked to physiological stress and could therefore reveal an individual's ability to cope with stressors.

Bayesian paternity analysis and mating patterns in a parasitic nematode, Trichostrongylus tenuis.

Mating behaviour is a fundamental aspect of the evolutionary ecology of sexually reproducing species, but one that has been under-researched in parasitic nematodes. We analysed mating behaviour in the parasitic nematode Trichostrongylus tenuis by performing a paternity analysis in a population from a single red grouse host. Paternity of the 150 larval offspring of 25 mothers (sampled from one of the two host caeca) was assigned among 294 candidate fathers (sampled from both caeca). Each candidate father's probability of paternity of each offspring was estimated from 10-locus microsatellite genotypes. Seventy-six (51%) offspring were assigned a father with a probability of >0.8, and the estimated number of unsampled males was 136 (95% credible interval (CI) 77-219). The probability of a male from one caecum fathering an offspring in the other caecum was estimated as 0.024 (95% CI 0.003-0.077), indicating that the junction of the caeca is a strong barrier to dispersal. Levels of promiscuity (defined as the probability of two of an adult's offspring sharing only one parent) were high for both sexes. Variance in male reproductive success was moderately high, possibly because of a combination of random mating and high variance in post-copulatory reproductive success. These results provide the first data on individual mating behaviour among parasitic nematodes.